The present invention provides nucleic acid sequences and methods to determine gene function. In particular, the present invention relates to reducing the levels of RNA encoded by a DNA sequence of interest using ribozymes. The present invention further relates to ribozyme sequences, recombinant expression vectors encoding ribozymes as well as host cells and transgenic animals comprising such expression vectors.
The human genome project and allied interests will soon have elucidated the sequence of the entire human genome [Cox et al (1994) Science 265:2031-2031; Guyer et al (1995) Proc. Natl. Acad. Sci. USA 92:10841-10848]. While this anticipated advance is exciting, it is also misleading since knowledge of the sequences of open reading frames and genetic coding regions, without a knowledge of the function of the gene products of this vast array of putative genes, provides only very limited insight into the human genome. Full knowledge of the genome requires knowledge of the function of each of the gene products of the putative genetic coding sequences. While gene function determination is ongoing within the field of molecular genetics, the rate at which the function of a gene can be determined is many orders of magnitude slower than the rate at which a gene can be sequenced. Therefore, a massive backlog of genetic sequences in search of a function looms on the horizon.
One of the traditionally used means of determining gene function is by xe2x80x9cknocking outxe2x80x9d or disrupting the coding sequence in an animal model and observing which structure(s) or function(s) is deleted in the resulting xe2x80x9cknock outxe2x80x9d model [Capecchi et al (1989) Science 244:1288-1292; Hasty et al (1991) Nature 350:243-246]. Gene knockouts are presently accomplished by homologous recombination in embryonic stem (ES) cells with a targeting vector, production of mosaic animals (in particular, mice) with a single disrupted allele in some of their germ cells, breeding to mice which are heterozygous for the disrupted gene, and finally inbreeding to venerate homozygous mice where the targeted gene disruption is present in each allele so that the full gene complement is rendered non-functional [Shastry et al (1994) Mol. Cellul. Biochem. 136:171-182; Galli-Taliadoros et al (1995) J. Immunol. Methods 181:1-15]. Only when each of these very time consuming steps has been successfully accomplished will a gene knockout mouse, which will show the phenotype associated with deletion of the targeted gene, be available for further determination of the function of the deleted gene. Clearly, this is a complex and slow method for gene function determination and cannot be expected, in a reasonable time, to yield the function of the many tens of thousands of coding sequences elucidated by the present flurry of genetic sequencing.
Therefore, there remains a need for a rapid and specific means for the determination of gene function.
The present invention provides methods for the identification of one or more functions of a nucleotide sequence in an organism which are useful for the rapid identification of, for example, disease related genes which may be targeted for the treatment or prevention of disease. The invention further provides recombinant expression vectors which encode ribozyme sequences, transgenic host cells, transgenic embryos and transgenic organisms which express a ribozyme sequence that is capable of cleaving mRNA encoded by the nucleotide sequence whose function is sought to be determined. The ribozyme sequences, recombinant expression vectors, transgenic host cells, transgenic embryos and transgenic organisms provided by the present invention are useful in identifying the function of any sequence of interest in an organism.
In one embodiment, the invention provides a ribozyme sequence capable of cleaving RNA, wherein the RNA is encoded by SEQ ID NO:5. While not restricted to any particular ribozyme sequence, in one preferred embodiment, the ribozyme sequence is selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21.
In another embodiment, the invention contemplates a DNA sequence encoding a ribozyme sequence capable of cleaving RNA encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:27. Although it is not intended that the DNA sequence be limited to any particular sequence, in one preferred embodiment, the DNA sequence is selected from the group consisting of DNA sequences encoding SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.
In yet another embodiment, the invention provides a recombinant expression vector comprising a DNA sequence encoding a ribozyme sequence capable of cleaving RNA encoded by SEQ ID NO:5. While not restricted to any ribozyme sequence, in a preferred embodiment, the ribozyme sequence is selected from the group consisting of SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21. In an alternative embodiment, it is preferred, though not required, that the recombinant expression vector is selected from the group consisting of pT7vaRz435, pT7vaRz365, and pT7vaRz564. In a particularly preferred embodiment, the recombinant expression vector is selected from the group consisting of pT7GaRz435, pT7GaRz365 and pT7GaRz564.
Also provided by the invention is a host cell comprising a recombinant expression vector wherein the recombinant expression vector comprises a DNA sequence encoding a ribozyme sequence capable of cleaving RNA encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:27. While not restricted to a particular host cell, one preferred embodiment contemplates that the host cell is a fertilized egg.
The invention further provides a transgenic zebrafish cell comprising a heterologous ribozyme sequence capable of cleaving an RNA sequence comprised in the zebrafish cell. In one preferred embodiment, the transgenic zebrafish cell is a fertilized egg. In an alternative embodiment, the transgenic zebrafish cell is an embryonic cell. In yet another alternative embodiment, the transgenic zebrafish cell is an adult cell.
The present invention further provides a method for identifying one or more functions of a DNA sequence of interest comprised in a genome of an organism, comprising: a) providing: i) a cell derived from the organism, wherein the cell comprises the DNA sequence of interest encoding RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; and c) detecting one or more changes in the manipulated cell relative to the derived cell thereby identifying one or more functions of the DNA sequence in the organism.
The present invention also provides a method for identifying one or more functions of a DNA sequence of interest comprised in a genome of an organism, comprising: a) providing: i) a cell derived from the organism, wherein the cell comprises the DNA sequence of interest encoding RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; c) permitting the manipulated cell to generate progeny cells; and d) detecting one or more changes in at least one of the progeny cells relative to the derived cell thereby identifying one or more functions of the DNA sequence of interest in the organism.
Also provided by the present invention is a method for identifying one or more functions of a first DNA sequence of interest comprised in a genome of a first organism, comprising: a) providing: i) a cell derived from a second organism, wherein the derived cell comprises a genome comprising a second DNA sequence homologous to the first DNA sequence, and wherein the second DNA sequence encodes RNA sequence; and ii) an expression vector comprising a third DNA sequence encoding a ribozyme sequence capable of cleaving a substrate cleavage sequence in the encoded RNA sequence, wherein the third DNA sequence is operably linked to a promoter sequence; b) introducing the expression vector into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the ribozyme sequence is expressed and wherein the encoded RNA sequence is cleaved by the expressed ribozyme sequence; c) permitting the manipulated cell to generate progeny cells; and d) detecting one or more changes in at least one of the progeny cells relative to the derived cells thereby identifying one or more functions for the first DNA sequence of interest in the first organism.
The present invention additionally a method for identifying one or more functions of a first DNA sequence comprised in a genome of a first organism, comprising: a) providing: i) a cell derived from a second organism, wherein the cell comprises a genome comprising a second DNA sequence homologous to the first DNA sequence; ii) a first expression vector comprising a DNA sequence encoding, a ribozyme sequence capable of cleaving a substrate cleavage sequence in the second DNA sequence wherein the DNA sequence is operably linked to a promoter sequence: and iii) a second expression vector comprising a coding sequence for an RNA polymerase operably linked to a cognate promoter of the RNA polymerase; iv) the RNA polymerase; b) introducing the first expression vector, the second expression vector, and the RNA polymerase into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the ribozyme sequence is expressed and wherein the second DNA sequence is cleaved by the expressed ribozyme sequence; c) permitting the manipulated cell to generate progeny cells; and d) detecting one or more changes in at least one of the progeny cells relative to the derived cell thereby identifying one or more functions for the first DNA sequence in the first organism.
The present invention also provides a method for identifying one or more functions of a first DNA sequence comprised in a genome of a first organism, comprising: a) providing: i) a cell derived from a second organism, wherein the derived cell comprises a genome comprising a second DNA sequence homologous to the first DNA sequence, and wherein the second DNA sequence encodes an RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; and c) detecting one or more changes in the manipulated cell relative to the derived cell thereby identifying at least one function for the first DNA sequence in the first organism.
While not limited to any particular organism, in one preferred embodiment, the first organism is human and the second organism is zebrafish. In an alternative preferred embodiment, the organism is human and the second organism is murine.
Although it is not contemplated that the ribozyme is restricted to any particular sequence, in one embodiment, the ribozyme is selected from the group consisting of group I intron ribozyme, ribonuclease P ribozyme, hammerhead ribozyme, hairpin ribozyme and hepatitis delta virus ribozyme. In a preferred embodiment, the ribozyme is a hammerhead ribozyme comprising a first substrate binding region, a second substrate binding region and a catalytic region, and wherein the first and second binding regions consist of 8 nucleotides.
While not intending to restrict the three-dimensional configuration of the second DNA sequence in which the substrate cleavage sequence is contained, in one embodiment, the substrate cleavage sequence is contained in a loop structure in the second DNA sequence. In a particularly preferred embodiment, the loop structure is selected from the group consisting of a 9-nucleotide loop, a 12-nucleotide loop and a 14-nucleotide loop.
Without intending to limit the promoter sequence to any particular sequence, in one preferred embodiment, the promoter sequence comprises an adenovirus type 2-associated RNA I gene promoter sequence. In a yet more preferred embodiment, the promoter sequence further comprises a promoter sequence selected from the group consisting of tRNA, CMV, RSV, SV40, PEPCK, MT, SRxcex1, P450 family, GAL7, T7, T3, SP6, K11 and heat shock protein promoter sequences. In an alternative preferred embodiment, the promoter sequence comprises a CMV promoter sequence, a T7 promoter sequence, and a vaRNA I promoter sequence.
Although it is not intended that the scope of the invention be limited to any particular cell type, in one preferred embodiment, the derived cell is an embryonic cell.
Additionally, it is contemplated that the methods of the invention are not limited to any particular type of expression. In one preferred embodiment, expression is transient. In an alternative preferred embodiment the expression is stable.
The invention further provides a method for identifying one or more functions of a first DNA sequence comprised in a genome of a first organism, comprising: a) providing: i) an oocyte derived from a second organism, wherein the derived oocyte comprises a genome comprising a second DNA sequence homologous to the first DNA sequence, and wherein the second DNA sequence encodes an RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived oocyte to generate a manipulated oocyte, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; and c) detecting one or more changes in the manipulated oocyte relative to the derived oocyte thereby identifying at least one function for the first DNA sequence in the first organism.
Also provided ny the present invention is a method for identifying one or more functions of a first DNA sequence comprised in a genome of a first organism, comprising: a) providing: i) a cell derived from a second organism, wherein the derived cell is transparent and comprises a genome comprising a second DNA sequence homologous to the first DNA sequence, and wherein the second DNA sequence encodes an RNA sequence; and ii) a ribozyme sequence capable of cleaving the RNA sequence; b) introducing the ribozyme sequence into the derived cell to generate a manipulated cell, wherein the introducing is under conditions such that the RNA sequence is cleaved by the ribozyme sequence; and c) detecting one or more changes in the manipulated cell relative to the derived cell thereby identifying at least one function for the first DNA sequence in the first organism.